The overall objective of this project is to understand how the unique substrate specificities of the mammalian microsomal cytochrome P450 enzymes arise. These enzymes participate in the metabolism of a wide range of drugs and environmental contaminants. Differences in P450 structure or expression can lead to inter-individual differences in drug metabolism that determine safe exposure limits for these compounds. Evolutionary divergence leads to functional differences between orthologous P450s that render extrapolation of animal studies to man imprecise. This underscores the importance of directly characterizing human P450s and of developing methods to predict drug metabolism. Mammalian P450s are membrane proteins, and this has limited the generation of crystals suitable for structural studies. Suitable modifications to the membrane binding domains of P450 2C5 reduced membrane association while promoting solubility and mono-dispersity. The modified P450 2C5 was crystallized leading to the determination of the first structure of a mammalian P450. The proposed studies will better characterize structural determinants of human drug metabolism by addressing three specific aims. (1) The approach taken to crystallize 2C5 will be extended to the human drug metabolizing P450s 2C9, 2D6 and 3A4. Additional modifications that may be necessary to produce soluble, active human P450s will be identified using chimeric proteins harboring alterations that allowed crystallization of 2C5. (2) The membrane topology of 2C5 will be better defined through the use of anti-peptide antibodies to identify portions of the microsomal membrane bound protein that are exposed. This information will be used as a design aid to address the first specific aim. (3) Models for the substrate specificity of human drug metabolizing enzymes will be generated based on the structure of 2C5. Hypotheses regarding determinants of substrate specificity will be tested by engineering 2C5 to mimic the activities of the human enzyme. These studies will increase our ability to predict human drug metabolism.